Proper DNA dosage has been recognized as essential for normal cellular development and function. A wide variety of specific DNA dosage defects are known, ranging from single gene duplication to gene amplification and partial chromosomal duplication to whole chromosomal additions, each of which typically results in a number of adverse consequences. For example, gene duplication and amplification have been positively linked to cellular oncogenic transformation, while chromosomal aneuploidies have been linked to serious birth defects.
One type of aneuploidy known to cause Down Syndrome is the trisomy of chromosome 21. Down Syndrome is one of the most common causes of mental retardation and is observed in approximately 1/800 live births. Although most Down Syndrome cases are due to an extra chromosome, partial duplications of a critical region on chromosome 21 in the DSCR (i.e. Down Syndrome Critical Region) can also cause clinical symptoms. Other trisomies compatible with life besides trisomy 21 include trisomy 13, trisomy 18, and various X and Y chromosome trisomies that lead to conditions such as Turner's Syndrome and Klinfelter's Syndrome.
The risk of child birth defects, including Down Syndrome, increases with a mother's age. To this end, prenatal detection of trisomies has been made available to women with advanced maternal age (over age 35). Moreover, defect testing is typically performed on pregnant women who have previously given birth to children with chromosomal abnormalities, and also to confirm fetus ultrasound image testing that indicates the possibility of Down Syndrome.
The conventional diagnosis of Down Syndrome is by chromosome karyotype. A karyotype shows the complete chromosome complement in a cell. Karyotypes detect any chromosomal aneuploidy (i.e. numerical abnormality) as well as structural chromosomal rearrangements. Sample types for chromosomal analysis include cultured amniotic cells or chorionic villi. Postnatal samples typically are peripheral whole blood. The strength of karyotype testing is a definitive diagnosis of aneuploidy and the ability to detect any chromosomal abnormality such as rearrangements and large chromosomal deletions and insertions. However, several drawbacks to the procedure exist, such as the necessity to culture cells, which in the case of prenatal diagnosis may take up to 2 weeks. Moreover, as the culturing process requires viable cells, it is unable to test a product of conception that has not yet reached certain developmental stages, such as an embryo or fetus. Further, karyotyping may not be able to detect small duplications in the Down's syndrome critical region.
One molecular cytogenetic method that has been used for faster detection of trisomies is the technique know as fluorescent in situ hybridization (FISH). FISH employs a system of fluorescent hybridization probes and antibodies to directly visualize the location of a target nucleotide sequence in a DNA molecule. See, In Situ Hybridization. A Practical Approach. Edited by D. G. Wilkinson, IRL Press, Oxford University Press (1994). While FISH does offer several advantages in the detection of trisomies over karyotyping, it is still quite time consuming, requires intact cells, a fluorescent microscope, and technical expertise in the use and operation of such equipment. Furthermore, FISH may not detect small duplications, such as those recited above in the DSCR of chromosome 21.
Another method for detecting trisomies has focused on the use of short tandem repeats (STRs). See, Findlay et al. Rapid Trisomy Diagnosis (21, 18, and 13) Using Fluorescent PCR and Short Tandem Repeats. Journal of Assistant Reproduction and Genetics. Vol. 15, No. 5. p 266–275. STRs are 2 to 5 nucleotides repeated a variable number of times. STRs are amplified by PCR and are separated by size on a sequencing gel. The band intensity is used to determine the number of copies of each allele. This technique, although suitable for simultaneous detection of a number of trisomies has not been widely implemented in clinical laboratories. Further, this techniques continues to suffer from a variety of the above recited disadvantages, as well as others, such as a limited number of STR loci compared to SNP loci on a chromosome.
A recent device that has been used for the quantification of gene dosage, as well as the identification of mutations and other genetic phenomena is marketed under the trade name LightCycler® by Roche Diagnostics, GMBH (Penzberg, Del.). Essentially, this instrument couples a thermocycler for polymerase chain reaction (PCR) amplification of materials with a fluorescence detector that allows for the real-time monitoring of amplification products. Specific examples of various instrument configurations, reagents for use therein, and uses therefor are found in U.S. Pat. Nos. 5,455,175, 5,935,522, 6,140,054, 6,174,670, 6,197,520,6,232,079, 6,254,514, 6,303,305, and 6,387,621, each of which is incorporated herein by reference. Further, specific methods of detecting certain DNA Duplications and Deletions using the LightCycler® have been disclosed by Ruiz-Ponte et al. in an article entitled Rapid Real-Time Fluorescent PCR Gene Dosage Test for the Diagnosis of DNA Duplications and Deletions, which is incorporated herein by reference. See, Ruiz-Ponte et al., Clinical Chemistry, 46:10, 1574–1582 (2000). Moreover, specific methods of detecting certain mutations using the LightCycler® is disclosed by Elaine Lyon in an article entitled Mutation Detection Using Fluorescent Hybridization Probes and Melting Curve Analysis, which is also incorporated herein by reference. See, Lyon, Exp. Rev. Mol. Diagn. 1 (1), (2001).
Despite the successful implementation of the above-recited methods and protocols for their respective purposes, the afore-mentioned disadvantages, as well as others, present needs that are not met by the current state of the art. Therefore, additional methods for the quantification of alleles and resultant detection of trisomic conditions, especially of chromosome 21, continue to be sought through ongoing research and development efforts.